Universal stem-loop primer method for screening and quantification of microRNA

RT-qPCR is the accepted technique for the quantification of microRNA (miR) expression: however, stem-loop RT-PCR, the most frequently used method for quantification of miRs, is time- and reagent-consuming as well as inconvenient for scanning. We established a new method called 'universal stem-l...

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Bibliographic Details
Published in:PloS one 2014-12, Vol.9 (12), p.e115293-e115293
Main Authors: Yang, Li-hong, Wang, Si-lu, Tang, Li-li, Liu, Biao, Ye, Wen-le, Wang, Ling-ling, Wang, Zhang-yang, Zhou, Meng-tao, Chen, Bi-cheng
Format: Article
Language:English
Subjects:
Acids
Adult
Biology and life sciences
Consent
Dissociation
DNA Primers - chemistry
DNA Primers - genetics
Experiments
Female
Gel electrophoresis
Gene expression
Humans
Hybridization
Immunocompromised Host
Kidney Transplantation
Laboratories
Lymphocytes
Lymphocytes T
Male
Medical research
Methods
MicroRNAs
MicroRNAs - blood
MicroRNAs - genetics
Middle Aged
miRNA
Nucleotide sequence
Nucleotides
Peripheral blood
Polymerase chain reaction
Primers
Pyridine nucleotide reductase
Research and Analysis Methods
Reverse Transcriptase Polymerase Chain Reaction - methods
Ribonucleic acid
RNA
Screening
Surgery
Test procedures
Thymic stromal lymphopoietin
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Summary:RT-qPCR is the accepted technique for the quantification of microRNA (miR) expression: however, stem-loop RT-PCR, the most frequently used method for quantification of miRs, is time- and reagent-consuming as well as inconvenient for scanning. We established a new method called 'universal stem-loop primer' (USLP) with 8 random nucleotides instead of a specific sequence at the 3' end of the traditional stem-loop primer (TSLP), for screening miR profile and to semi-quantify expression of miRs. Peripheral blood samples were cultured with phytohaemagglutinin (PHA), and then 87 candidate miRs were scanned in cultured T cells. By USLP, our study revealed that the expression of miR-150-5p (miR-150) decreased nearly 10-fold, and miR-155-5p (miR-155) increased more than 7-fold after treated with PHA. The results of the dissociation curve and gel electrophoresis showed that the PCR production of the USLP and TSLP were specificity. The USLP method has high precision because of its low ICV (ICV
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0115293